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Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease.

Bender T, Lewrenz I, Franken S, Baitzel C, Voos W - Mol. Biol. Cell (2011)

Bottom Line: To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins.Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems.Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie (IBMB), Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.

ABSTRACT
Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

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Identification of aggregated mitochondrial proteins. (A) Coomassie stain of an SDS gel with lysates of isolated mitochondria from S. cerevisae after treatment at the indicated temperatures and sedimentation of aggregates by ultracentrifugartion at 125,000 × g. Protein bands whose intensity increased with rising incubation temperature are marked with an asterisk (*); those whose intensity did not change with temperature are marked with #. T, total; Sup, supernatant; Pel, pellet. (B) Aggregates after treatments at 25°C or 42°C were spun down at 125,000 × g, and the indicated bands identified by mass spectrometry. (C) Quantitative analysis of mitochondrial protein aggregation on 2D-PAGE. Residual spot volume intensities were determined in the soluble fraction after heat treatment and a high-velocity spin and compared with intensities in total mitochondrial lysates. The relative difference of spot intensities in supernatant vs. total (set to 100%) for individual protein species are shown as means of three independent experiments.
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Figure 1: Identification of aggregated mitochondrial proteins. (A) Coomassie stain of an SDS gel with lysates of isolated mitochondria from S. cerevisae after treatment at the indicated temperatures and sedimentation of aggregates by ultracentrifugartion at 125,000 × g. Protein bands whose intensity increased with rising incubation temperature are marked with an asterisk (*); those whose intensity did not change with temperature are marked with #. T, total; Sup, supernatant; Pel, pellet. (B) Aggregates after treatments at 25°C or 42°C were spun down at 125,000 × g, and the indicated bands identified by mass spectrometry. (C) Quantitative analysis of mitochondrial protein aggregation on 2D-PAGE. Residual spot volume intensities were determined in the soluble fraction after heat treatment and a high-velocity spin and compared with intensities in total mitochondrial lysates. The relative difference of spot intensities in supernatant vs. total (set to 100%) for individual protein species are shown as means of three independent experiments.

Mentions: To determine the extent of protein aggregation in an in vivo–like situation, we subjected isolated mitochondria from S. cerevisiae to a 20-min heat stress treatment. Although a temperature of 30°C can be considered physiological, an increase to 37°C or 42°C represents a mild or strong heat shock, respectively. During the stress treatment, mitochondria were supplied with ATP and NADH to keep them fully energized. Separation of aggregated proteins by a high-velocity centrifugation after in vivo heat stress and subsequent detergent lysis is a well-established method that has been used before to characterize protein aggregation in bacteria (Mogk et al., 1999). We used 0.5% Triton X-100 for lysis, which is a mild nonionic detergent that solubilizes lipid membranes but does not denature proteins. Lysates of the treated mitochondria were subjected to centrifugation at 125,000 × g in order to separate soluble proteins, which remain in the supernatant, from aggregated polypeptides that sediment to the pellet fraction. After analysis of the pellet fraction by SDS–PAGE, we observed a temperature-dependent increase in the protein amount for at least six protein bands (Figure 1A, marked with *). These bands presumably represent heat-labile proteins that were present in the soluble fraction at physiological temperatures (25°C or 30°C) but denatured during the stress treatment and formed insoluble aggregates. Most aggregating mitochondrial proteins were in the high-molecular weight range above 50 kDa. On the other hand, a certain set of proteins was found in the pellet fraction independently of the temperature at which the mitochondria were treated (Figure 1A, marked with #). We considered the possibility that some integral membrane proteins might sediment in our aggregation assay due to insufficient solubilization of membranes by detergent lysis. However, immunodecorations with a specific antiserum revealed that the abundant integral membrane protein ADP/ATP-carrier (AAC) is not present in the pellet under all temperature conditions tested (see Figure 2A), so a contamination with membrane proteins can be ruled out. We therefore reasoned that extremely large soluble protein complexes, for example, ribosomes, might also be sedimented under the centrifugation conditions applied. Due to their size and density, they should be found in the pellet independently of the temperature conditions. This could be confirmed by Western blotting and immunodecoration with antiserum directed against the mitochondrial ribosomal protein Mrpl40 that was present in the pellet fraction in similar amounts at all temperatures (see Figure 2A).


Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease.

Bender T, Lewrenz I, Franken S, Baitzel C, Voos W - Mol. Biol. Cell (2011)

Identification of aggregated mitochondrial proteins. (A) Coomassie stain of an SDS gel with lysates of isolated mitochondria from S. cerevisae after treatment at the indicated temperatures and sedimentation of aggregates by ultracentrifugartion at 125,000 × g. Protein bands whose intensity increased with rising incubation temperature are marked with an asterisk (*); those whose intensity did not change with temperature are marked with #. T, total; Sup, supernatant; Pel, pellet. (B) Aggregates after treatments at 25°C or 42°C were spun down at 125,000 × g, and the indicated bands identified by mass spectrometry. (C) Quantitative analysis of mitochondrial protein aggregation on 2D-PAGE. Residual spot volume intensities were determined in the soluble fraction after heat treatment and a high-velocity spin and compared with intensities in total mitochondrial lysates. The relative difference of spot intensities in supernatant vs. total (set to 100%) for individual protein species are shown as means of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046053&req=5

Figure 1: Identification of aggregated mitochondrial proteins. (A) Coomassie stain of an SDS gel with lysates of isolated mitochondria from S. cerevisae after treatment at the indicated temperatures and sedimentation of aggregates by ultracentrifugartion at 125,000 × g. Protein bands whose intensity increased with rising incubation temperature are marked with an asterisk (*); those whose intensity did not change with temperature are marked with #. T, total; Sup, supernatant; Pel, pellet. (B) Aggregates after treatments at 25°C or 42°C were spun down at 125,000 × g, and the indicated bands identified by mass spectrometry. (C) Quantitative analysis of mitochondrial protein aggregation on 2D-PAGE. Residual spot volume intensities were determined in the soluble fraction after heat treatment and a high-velocity spin and compared with intensities in total mitochondrial lysates. The relative difference of spot intensities in supernatant vs. total (set to 100%) for individual protein species are shown as means of three independent experiments.
Mentions: To determine the extent of protein aggregation in an in vivo–like situation, we subjected isolated mitochondria from S. cerevisiae to a 20-min heat stress treatment. Although a temperature of 30°C can be considered physiological, an increase to 37°C or 42°C represents a mild or strong heat shock, respectively. During the stress treatment, mitochondria were supplied with ATP and NADH to keep them fully energized. Separation of aggregated proteins by a high-velocity centrifugation after in vivo heat stress and subsequent detergent lysis is a well-established method that has been used before to characterize protein aggregation in bacteria (Mogk et al., 1999). We used 0.5% Triton X-100 for lysis, which is a mild nonionic detergent that solubilizes lipid membranes but does not denature proteins. Lysates of the treated mitochondria were subjected to centrifugation at 125,000 × g in order to separate soluble proteins, which remain in the supernatant, from aggregated polypeptides that sediment to the pellet fraction. After analysis of the pellet fraction by SDS–PAGE, we observed a temperature-dependent increase in the protein amount for at least six protein bands (Figure 1A, marked with *). These bands presumably represent heat-labile proteins that were present in the soluble fraction at physiological temperatures (25°C or 30°C) but denatured during the stress treatment and formed insoluble aggregates. Most aggregating mitochondrial proteins were in the high-molecular weight range above 50 kDa. On the other hand, a certain set of proteins was found in the pellet fraction independently of the temperature at which the mitochondria were treated (Figure 1A, marked with #). We considered the possibility that some integral membrane proteins might sediment in our aggregation assay due to insufficient solubilization of membranes by detergent lysis. However, immunodecorations with a specific antiserum revealed that the abundant integral membrane protein ADP/ATP-carrier (AAC) is not present in the pellet under all temperature conditions tested (see Figure 2A), so a contamination with membrane proteins can be ruled out. We therefore reasoned that extremely large soluble protein complexes, for example, ribosomes, might also be sedimented under the centrifugation conditions applied. Due to their size and density, they should be found in the pellet independently of the temperature conditions. This could be confirmed by Western blotting and immunodecoration with antiserum directed against the mitochondrial ribosomal protein Mrpl40 that was present in the pellet fraction in similar amounts at all temperatures (see Figure 2A).

Bottom Line: To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins.Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems.Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie (IBMB), Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.

ABSTRACT
Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

Show MeSH
Related in: MedlinePlus